Performance of Daphnia magna on flour, leaves, and pollen from different maize lines: Implications for risk assessment of genetically engineered crops.

Non-target effects of genetically engineered (GE) plants on aquatic Daphnia magna have been studied by feeding the species with different maize materials containing insecticidal Cry proteins from Bacillus thuringiensis (Bt). The results of those studies were often difficult to interpret, because only one GE plant was compared to one related non-GE control. In such a setting, effects of the Cry proteins cannot be distinguished from plant background effects, in particular when the test species is nutritionally stressed. In the present study, we tested the suitability of three different maize materials, i.e., flour, leaves and pollen, from five diverse non-GE maize lines (including EXP 258, a breeding line that is closely related to a SmartStax Bt maize) as exclusive food sources for D. magna. The parameters recorded included survival, sublethal endpoints such as body size, number of moltings to first offspring, time to first offspring, number of individuals in first clutch, total number of clutches, total number of offspring, average number of offspring per clutch, and population measures such as net reproductive rate R0, generation time T and intrinsic rate of increase rm. The results showed that D. magna can survive, grow and reproduce when fed only maize materials, although the performance was poorer than when fed algae, which indicates nutritional stress. Large differences in life table and population parameters of D. magna were observed among the different maize lines. Our results suggest that confounding effects caused by nutritional stress and plant background might explain some of the conflicting results previously published on the effects of Bt crops on D. magna. Using 95% confidence intervals for the means of the five maize lines for all measured parameters of D. magna performance in our study, we captured the natural range of variation. This information is useful for the interpretation of observed differences in D. magna performance between a GE plant and its non-GE comparator as it helps judging whether observed effects are of biological relevance. If differences between a GE and comparator line are observed and their biological relevance needs to be assessed in future risk assessments of GE maize, 1) the data on natural variation of the different parameters generated by previous studies can be informative (e.g. data from our study for maize fed D. magna); 2) for additional experiments the inclusion of multiple unrelated non-GE comparators should be considered; In addition, it should be taken into account that nutritional stress can affect the outcome of the study.

Bacillus thuringiensis Maize Expressing a Fusion Gene Cry1Ab/Cry1AcZM Does Not Harm Valued Pollen Feeders.

The ladybird Propylea japonica, adults of the green lacewing Chrysoperla nipponensis and the honey bee Apis mellifera are common pollen feeders in many crop systems. They could therefore be directly exposed to Cry proteins in Bacillus thuringiensis (Bt)-transgenic crop fields by ingestion of pollen. They, or closely related species, are therefore often selected as surrogate test species in non-target risk assessment of Bt plants. In the current study, we evaluated the potential effects of the ingestion of Bt maize pollen containing the Cry1Ab/Cry1Ac fusion protein on various life-table parameters of the three pollen-feeding non-target species in laboratory feeding assays. The results showed that pupation rate and male adult fresh weight of P. japonica were significantly increased when fed pollen from Bt maize compared to control maize pollen, but other test life-table parameters were not affected. For the other two species, none of the tested life-table parameters (survival, pre-oviposition period, fecundity and adult fresh weight for C. nipponensis; survival and mean acinus diameter of hypopharyngeal glands for A. mellifera) differed between non-Bt and Bt maize pollen treatments. ELISA measurements confirmed the stability and uptake of the Cry protein by all three species during the feeding bioassays. In addition, a sensitive insect bioassay confirmed the bioactivity of the Cry1Ab/Cry1Ac protein in the Bt maize pollen used. Overall, the results suggested that the three pollen feeders are not sensitive to the Cry1Ab/Cry1Ac protein, and planting of the Bt maize variety will pose a negligible risk to P. japonica, adult C. nipponensis and adult A. mellifera.

The food safety of DP-356Ø43 soybeans on SD rats reflected by physiological variables and fecal microbiota during a 90-day feeding study.

Soybean is an important food resource for the eastern countries and herbicide-tolerant genetically modified soybeans (GMS) were widely developed to deal with weeds problems. Unprocessed soybean flour instead of dehulled and defatted soybean meal was used to reflect the safety of soybean food in whole. Rats were given formulated diets containing DP-356Ø43 or non-GM soybean JACK at an incorporation rate of 7.5%, 15%, or 30% (w/w), respectively for 90 days. Targeted traditional toxicological response variables were measured to reflect the holistic health of animals. No treatment-related adverse or toxic effects were observed based on an examination of the daily clinical signs, body weight, food consumption, hematology, serum biochemistry, and organ weight or based on gross and histopathological examination. The results demonstrate that the soybean DP-356Ø43 is as safe for consumption as conventional soybean JACK. In the current study, the effect of a herbicide-tolerant GMS DP-356043 on identified intestinal microbiota was evaluated in a rodent feeding study compared with its conventional control JACK. Feces samples from rats consuming different diets were collected before the start of the experiment (time 0) and at monthly intervals (at the end of the 1st, 2nd and 3rd months) over the course of 90 days. Six types of bacterias shared by humans and rats were detected with Q-PCR. The results of QPCR indicated that the GMS 356Ø43 had a comparable effect on the abundance of Bifidobacterium group, Clostridium perfringens subgroup, Escherichia coli, and Bacteroides-Prevotella group as the non-GMS JACK.

Subchronic toxicity study in rats evaluating genetically modified DAS-81419-2 soybean.

A 90-day feeding study in rats was conducted to evaluate the subchronic oral toxicity of genetically modified (GM) DAS-81419-2 soybean. Wistar rats were fed with diets containing toasted soybean meal produced from DAS-81419-2 soybean grain that expresses the Cry1F, Cry1Ac, and Pat proteins or containing conventional soybean at doses of 30.0%, 15.0%, 7.5%, or 0% (control group) for 90 consecutive days. The general behavior, body weight and food consumption were observed. At the middle and end of the experiment, blood, serum, and urine samples were collected for biochemical assays. At the conclusion of the study, the internal organs were weighed and histopathological examination was completed. The rats exhibited free movement and shiny coats without any abnormal symptoms or abnormal secretions in their noses, eyes, or mouths. There were no adverse effects on body weight in GM soybean groups and conventional soybean groups. No biological differences in hematological, biochemical, or urine indices were observed. No significant differences in relative organ weights were detected between the experimental groups and the control group. No histopathological changes were observed. Under the conditions of this study, DAS-81419-2 soybean did not cause any treatment-related effects in Wistar rats following 90 days of dietary administration.

Safety assessment of plant varieties using transcriptomics profiling and a one-class classifier.

An important part of the current hazard identification of novel plant varieties is comparative targeted analysis of the novel and reference varieties. Comparative analysis will become much more informative with unbiased analytical approaches, e.g. omics profiling. Data analysis estimating the similarity of new varieties to a reference baseline class of known safe varieties would subsequently greatly facilitate hazard identification. Further biological and eventually toxicological analysis would then only be necessary for varieties that fall outside this reference class. For this purpose, a one-class classifier tool was explored to assess and classify transcriptome profiles of potato (Solanum tuberosum) varieties in a model study. Profiles of six different varieties, two locations of growth, two year of harvest and including biological and technical replication were used to build the model. Two scenarios were applied representing evaluation of a 'different' variety and a 'similar' variety. Within the model higher class distances resulted for the 'different' test set compared with the 'similar' test set. The present study may contribute to a more global hazard identification of novel plant varieties.

Environmental risk assessment for the small tortoiseshell Aglais urticae and a stacked Bt-maize with combined resistances against Lepidoptera and Chrysomelidae in central European agrarian landscapes.

The cultivation of Lepidoptera-resistant Bt-maize may affect nontarget butterflies. We assessed the risk posed by event MON89034 × MON88017 (expressing Cry1A.105 and Cry2Ab2 against corn borers) to nontarget Lepidoptera. Using the small tortoiseshell Aglais urticae, a butterfly species common in central Europe, as a test organism we (i) assessed the toxicity of Bt-maize pollen on butterfly larvae; (ii) measured pollen deposition on leaves of the host plant Urtica dioica; (iii) mapped the occurrence and distribution of host plants and larvae in two arable landscapes in Germany during maize anthesis; and (iv) described the temporal occurrence of a 1-year population of A. urticae. (i) Larvae-fed 200 Bt-maize pollen grains/cm(2) had a reduced feeding activity. Significant differences in developmental time existed at pollen densities of 300 Bt-maize pollen grains/cm(2) and in survival at 400 grains/cm(2). (ii) The highest pollen amount found was 212 grains/cm(2) at the field margin. Mean densities were much lower. (iii) In one region, over 50% of A. urticae nests were located within 5 m of a maize field, while in the other, all nests were found in more than 25 m distance to a maize field. (iv) The percentage of larvae developing during maize anthesis was 19% in the study area. The amount of pollen from maize MON89034 × MON88017 found on host plants is unlikely to adversely affect a significant proportion of larvae of A. urticae. This paper concludes that the risk of event MON89034 × MON88017 to populations of this species is negligible.

Compositional and toxicological analysis of a GM potato line with reduced α-solanine content--a 90-day feeding study in the Syrian Golden hamster.

Steroidal glycoalkaloids (GAs) are toxins, produced by plants of the Solanaceae family. The potato plant (Solanum tuberosum L.) and its tubers predominantly contain the two GAs α-chaconine and α-solanine. These compounds are believed to act in synergy, and the degree of toxicity may therefore depend on their ratio in the potato. To determine the influence of α-solanine: α-chaconine ratio in potatoes on toxicity, a GM potato line (SGT 9-2) with reduced α-solanine content, and the parental control line (Desirée wild-type) having a traditional α-solanine: α-chaconine ratio were (1) studied for compositional similarity by analysing for a range of potato constituents, and (2) used in a 90-day feeding trial with the Syrian Golden hamster to study differential toxicity. The animal feeding study used diets with up to 60% freeze-dried potato powder from either line. Whilst data indicated some compositional differences between the GM line and its wildtype control these did not raise concerns related to nutritional value or safety. Results of the feeding trials showed a low number of significant differences between potato lines with different α-solanine: α-chaconine ratio but none were considered to raise safety concerns with regard to human (or animal) consumption.

[Nutritional components and sub-chronic toxicity of genetically modified rice expressing human lactoferrin].

OBJECTIVE: To compare the nutritional components of genetically modified rice expressing human lactoferrin (hLf) with its parental rice, and to observe the sub-chronic toxicity of hLf rice. METHODS: The nutritional components of hLf rice and its parental rice were determined by the National Standard Methods. Eighty weanling Wistar rats were randomly divided into 4 groups based on their gender and body weight: group A (hLf rice high-dose group with 71.45% rice), group B (hLf rice medium-dose group with 35. 725% rice), group C (parental rice group with 71.01% rice) and group D (AIN-93G diet group), and the latter two groups were used as the control. Body weight, dietary intake, blood routine test, blood biochemical examination, organ coefficient, bone density and the pathology of organs were investigated at the end of a 90-day feeding experiment. RESULTS: Except for human lactoferrin and Fe, there was no difference of main nutritional components, minerals and vitamins between groups. The differences of some indicators of blood routine (WBC, HGB, RBC and MCH), blood biochemistry (AST and GLU), organ coefficient and bone density between group A and B (hLf rice) with group C (parental rice) or group D (AIN-93G) were significant, while no difference of other indicators. CONCLUSION: Although some differences were observed, all indicators were still in the normal reference range. Therefore, there was no sign of toxic and adverse effects for hLf rice on rats.

A literature review on the safety assessment of genetically modified plants.

In recent years, there has been a notable concern on the safety of genetically modified (GM) foods/plants, an important and complex area of research, which demands rigorous standards. Diverse groups including consumers and environmental Non Governmental Organizations (NGO) have suggested that all GM foods/plants should be subjected to long-term animal feeding studies before approval for human consumption. In 2000 and 2006, we reviewed the information published in international scientific journals, noting that the number of references concerning human and animal toxicological/health risks studies on GM foods/plants was very limited. The main goal of the present review was to assess the current state-of-the-art regarding the potential adverse effects/safety assessment of GM plants for human consumption. The number of citations found in databases (PubMed and Scopus) has dramatically increased since 2006. However, new information on products such as potatoes, cucumber, peas or tomatoes, among others was not available. Corn/maize, rice, and soybeans were included in the present review. An equilibrium in the number research groups suggesting, on the basis of their studies, that a number of varieties of GM products (mainly maize and soybeans) are as safe and nutritious as the respective conventional non-GM plant, and those raising still serious concerns, was currently observed. Nevertheless, it should be noted that most of these studies have been conducted by biotechnology companies responsible of commercializing these GM plants. These findings suggest a notable advance in comparison with the lack of studies published in recent years in scientific journals by those companies. All this recent information is herein critically reviewed.

The oral toxicity of the transgenic Bt+CpTI cotton pollen to honeybees (Apis mellifera).

Transgenic insect-resistant cotton has been planted in China in a large scale and may have adverse impacts on honeybees. Pollens from the transgenic Cry1Ac+CpTI cotton Zhong-41 and the parental cotton Zhong-23 were collected from the field and their impacts on adult worker bees were assessed. Experimental results showed that Zhong-41 pollen had no acute oral toxic effect on worker bees. No significant differences were observed in the superoxide dismutase activity or in the longevity of worker bees fed with diets containing the two cotton pollens. The main reasons for the outcome may be the low expression level of the transgenic proteins Cry1Ac and CpTI in the pollen of Zhong-41 as well as the substantial equivalence in the amounts of gross protein and soluble saccharides for the two cotton pollens. The implications of these results are discussed and further work to be carried out is put forward.

Toxicity studies of genetically modified plants: a review of the published literature.

According to the information reported by the WHO, the genetically modified (GM) products that are currently on the international market have all passed risk assessments conducted by national authorities. These assessments have not indicated any risk to human health. In spite of this clear statement, it is quite amazing to note that the review articles published in international scientific journals during the current decade did not find, or the number was particularly small, references concerning human and animal toxicological/health risks studies on GM foods. In this paper, the scientific information concerning the potential toxicity of GM/transgenic plants using the Medline database is reviewed. Studies about the safety of the potential use of potatoes, corn, soybeans, rice, cucumber, tomatoes, sweet pepper, peas, and canola plants for food and feed were included. The number of references was surprisingly limited. Moreover, most published studies were not performed by the biotechnology companies that produce these products. This review can be concluded raising the following question: where is the scientific evidence showing that GM plants/food are toxicologically safe?

Toxins in transgenic crop byproducts may affect headwater stream ecosystems.

Corn (Zea mays L.) that has been genetically engineered to produce the Cry1Ab protein (Bt corn) is resistant to lepidopteran pests. Bt corn is widely planted in the midwestern United States, often adjacent to headwater streams. We show that corn byproducts, such as pollen and detritus, enter headwater streams and are subject to storage, consumption, and transport to downstream water bodies. Laboratory feeding trials showed that consumption of Bt corn byproducts reduced growth and increased mortality of nontarget stream insects. Stream insects are important prey for aquatic and riparian predators, and widespread planting of Bt crops has unexpected ecosystem-scale consequences.

Evaluation of the safety and nutritional equivalence of a genetically modified cottonseed meal in a 90-day dietary toxicity study in rats.

Meal prepared from Cry1F/Cry1Ac transgenic/genetically modified cottonseed (WIDESTRIKE Insect Protection, hereafter referred to as WIDESTRIKE) was compared to cottonseed meal prepared from four conventionally bred lines of cotton (three commercial non-transgenic line controls (PHY72, PHY78 and 98M-2983), and a near isoline non-transgenic control (PSC355) in a 90-day dietary study to evaluate safety and nutritional equivalence. Diets were formulated with 10% WIDESTRIKE cottonseed meal equivalent to 7,235 mg/kg/day for males and 7,935 mg/kg/day for females. Animals were evaluated by cage-side and hand-held detailed clinical observations, body weight, and feed consumption. Functional tests, motor activity and ophthalmic examinations were conducted pre-exposure and prior to study termination. Standard hematology, clinical chemistry, prothrombin time and urinalysis parameters were evaluated. All rats had a complete necropsy and selected organs were weighed. Histopathologic examinations were performed on all rats fed the diets containing the near isoline non-transgenic control or WIDESTRIKE. Following 90 days of feeding, no adverse effects were observed during the conduct of clinical observations or in any of the parameters measured in this study. This study demonstrated that rodent diets prepared with 10% cottonseed meal from WIDESTRIKE cottonseeds do not produce any untoward effects and are nutritionally equivalent to cottonseed meals prepared from other, non-transgenic cottonseeds.

A 90-day safety study in Wistar rats fed genetically modified rice expressing snowdrop lectin Galanthus nivalis (GNA).

Genetically modified plants expressing insecticidal traits offer a new strategy for crop protection, but at the same time present a challenge in terms of food safety assessment. The present 90-day feeding study was designed to assess the safety of a rice variety expressing the snowdrop Galanthus nivalis lectin (GNA lectin), and forms part of a EU-funded project where the objective has been to develop and validate sensitive and specific methods to assess the safety of genetically modified foods. Male and female Wistar rats were given a purified diet containing either 60% genetically modified or parental rice for 90 days. This corresponds to a mean daily GNA lectin intake of approximately 58 and 67mg/kg body weight for males and females, respectively. Prior to the animal study comprehensive analytical characterization of both rice materials was performed. The chemical analyses showed a number of statistically significant differences, with the majority being within the ranges reported in the literature. In the animal study a range of clinical, biological, immunological, microbiological and pathological parameters were examined. A number of significant differences were seen between groups fed the two diets, but none of them were considered to be adverse. In conclusion, the design of the present animal study did not enable us to conclude on the safety of the GM food. Additional group(s) where the expressed gene products have been spiked to the diet should be included in order to be able to distinguish whether the observed effects were due to the GNA lectin per se or to secondary changes in the GM rice.

Multigeneration reproductive and developmental toxicity study of bar gene inserted into genetically modified potato on rats.

Each specific protein has an individual gene encoding it, and a foreign gene introduced to a plant can be used to synthesize a new protein. The identification of potential reproductive and developmental toxicity from novel proteins produced by genetically modified (GM) crops is a difficult task. A science-based risk assessment is needed in order to use GM crops as a conventional foodstuff. In this study, the specific characteristics of GM food and low-level chronic exposure were examined using a five-generation animal study. In each generation, rats were fed a solid pellet containing 5% GM potato and non-GM potato for 10 wk prior to mating in order to assess the potential reproductive and developmental toxic effects. In the multigeneration animal study, there were no GM potato-related changes in body weight, food consumption, reproductive performance, and organ weight. Polymerase chain reaction (PCR) was carried out using extracted genomic DNA to examine the possibility of gene persistence in the organ tissues after a long-term exposure to low levels of GM feed. In each generation, the gene responsible for bar was not found in any of the reproductive organs of the GM potato-treated male and female rats, and the litter-related indexes did not show any genetically modified organism (GMO)-related changes. The results suggest that genetically modified crops have no adverse effects on the multigeneration reproductive-developmental ability.

Will transgenic plants adversely affect the environment?

Transgenic insecticidal plants based on Bacillus thuringiensis (Bt) endotoxins, on proteinase inhibitors and on lectins, and transgenic herbicide tolerant plants are widely used in modern agriculture. The results of the studies on likelihood and non-likelihood of adverse effects of transgenic plants on the environment including: (i) effects on nontarget species; (ii) invasiveness; (iii) potential for transgenes to 'escape' into the environment by horizontal gene transfer; and (iv) adverse effects on soil biota are reviewed. In general, it seems that large-scale implementation of transgenic insecticidal and herbicide tolerant plants do not display considerable negative effects on the environments and, moreover, at least some transgenic plants can improve the corresponding environments and human health because their production considerably reduces the load of chemical insecticides and herbicides.